Interaction of RBP (retinol binding protein) with prealbumin. Under physiological conditions, the retinol binding protein (RBP) forms a stable complex with the protein prealbumin. According to binding studies conducted in other laboratories, 1 prealbumin molecule binds to 1 molecule of RBP at neutral pH in a buffer containing 0.1M NaCl. The dissociation constant of the complex has been estimated to be 10 to the minus 7th power M. In my laboratory we have attempted to use fluorescence spectroscopy as a tool to evaluate the dissociation constant. The retinol chromophore (all-trans) binds to the retinol binding protein with a dissociation constant of 10 to the minus 8th power M. This binding process is parallelled by changes in the spectroscopic properties of retinol. Thus, the fluorescence lifetime is enhanced (8 nanoseconds) and the emission anisotropy (A equals 0.30) is shorter than the limiting emission anisotropy (A equals 0.37) detected in rigid glasses. Addition of prealbumin to the retinol binding protein at a mixing ratio of 1:1 does not influence the degree of emission anisotropy. Furthermore, the fluorescence lifetime of retinol remains constant (8 nanoseconds). Although the technique of fluorescence depolarization could not be used to measure the stoichiometry of binding, we were able to detect the strong interaction between RBP and prealbumin by means of nanosecond emission techniques. The RBP is characterized by a rotational correlation time of 8 ns at pH 7, 25 degrees C. Upon addition of prealbumin (10 to the minus 6th power M), mixing ratio 1:1, the rotational correlation time is increased to 26 nanoseconds. The increment is obviously due to the association of prealbumin (50.000) with RBP (21.000). Further addition of RBP has no effect on the longest rotational correlation time detected by emission anisotropy measurements.